Not Applicable
The present invention relates generally to a method for improving the calibration of a mass spectrometer. More specifically, the invention is a method whereby a mass spectra generated by a mass spectrometer is calibrated by shifting the parameters used by the spectrometer to assign masses to the spectra in a manner which reconciles the signal of ions within the spectra having equal mass but differing charge states, or by reconciling ions having known differences in mass to relative values consistent with those known differences. In this manner, the present invention allows calibration of the mass spectrometer without the need for standards while allowing the generation of a highly accurate mass spectra by the instrument.
The ability of mass spectrometry to rapidly sort through complex biological mixtures and identify the component proteins, peptides, oligonucleo-tides, and noncovalent complexes is rapidly being adopted in biological research, especially for proteome characterization and protein profiling. There is a well recognized need for the high throughput identification of these and other species, for example proteins and their posttranslational modifications that are, for example, up-regulated or down-regulated in response to a specific external stimulus, the onset of disease, or normal aging. The conventional approach to proteomics involves the high resolution separation of proteins using 2D polyacryl-amide gel electrophoresis followed by their one-at-a-time excision and characterization, increasingly exploiting mass spectrometry. Additional information is generally gathered in the form of a correlation between the peptide masses for peptide fingerprinting (e.g., their common origin from a single protein), or by partial peptide sequencing. However, even complete automation of separations and sample processing imposes practical limitations upon the throughput of these methods.
The use of higher mass accuracy mass measurements has the potential to greatly speed proteome characterization and protein identification. Sufficiently high mass measurement accuracy, in principal, can enable the identification of a protein from a single peptide mass. Thus, a complex protein mixture can be enzymatically digested and the resulting peptide mixture separated and used for protein profiling and posttranslational modification determination. Yates and co-workers have pioneered an approach based upon capillary liquid chromatography tandem mass spectrometry (LC-MS/MS) of enzymatically digested protein mixtures in McCormack, A. L.; Schieltz, D. M.; Goode, B.; Yang, S.; Barnes, G.; Drubin, D.; Yates, J. R. Anal. Chem. 1997, 69, 767-776, the entire contents of which is incorporated herein by this reference.
Processing of more complex mixtures for ever higher throughput analyses, such as the analysis of whole proteomes, results in much greater demands on mass spectrometry, in terms of speed, resolution, mass measurement accuracy, and data-dependent acquisition. As such, calibration schemes that can enable higher mass accuracy measurements to be accomplished over a wide range of conditions play an essential role in the successful application of mass spectrometry to protein identification from complex peptide mixtures. Experiments involving on-line chromatographic or electrophoretic separations also present the additional constraint that mass calibration functions, for example, in Fourier transform ion cyclotron resonance (FTICR), can change from spectrum to spectrum for reasons related to variations in the size of the trapped ion population. For example, Easterling et al. recently demonstrated that the detected cyclotron frequency (and the derived mass measurement) in FTICR experiments could change over a range of 110 ppm for MALDI mass spectra of the peptide bradykinin de-pending upon trapped ion population size in Easterling, M. L.; Mize, T. H.; Amster, I. J. Anal. Chem. 1999, 71, 624-632, the entire contents of which are incorporated herein by this reference. Clearly, such a level of mass measurement uncertainty greatly limits protein characterization efforts and generally precludes the use of mass measurements for single peptide species for protein identification (i.e., to serve as a xe2x80x9cbiomarkerxe2x80x9d for a specific protein). Importantly, Easterling et al. also showed that this frequency shift, at least to the very low ppm level is linearly related to the number of trapped ions and thus, can be effectively corrected when the ion population size is known or reproducibly controlled. This observed effect of ion population is also consistent with the understanding of the effects of space charge upon ion cyclotron motion in FTICR. In Burton, R. D.; Matuszak, K. P.; Watson, C. H.; Eyler, J. R. J. Am. Soc. Mass Spectrom. 1999, 10, 1291-1297, the entire contents of which are incorporated herein by this reference, Burton et al. showed that measurements based upon xe2x80x9cexternal calibrationxe2x80x9d and a single xe2x80x9cinternalxe2x80x9d standard could provide mass accuracies essentially equivalent to those obtained with multiple internal calibrants, and an order of magnitude greater accuracy than external calibration alone. These results are also consistent with the conclusion of Easterling et al., showing that variations in trapped ion population sizes lead to essentially constant ion cyclotron frequency shifts or offsets across the mass spectrum.
Space-charge effects on mass calibration are manifested by stepwise shifts, or offsets, of all frequencies to an extent that depends upon ion population size, a quantity that is generally unknown or not well defined in most experiments. Thus, the requirement for prior knowledge of the sample, the trapped ion population, or the conditions under which the measurements were made, presents a drawback for this technique, and there is still a general need for improved methods for calibrating a mass spectometer without the use of calibrants and where the ion population size is unknown.
The present invention exploits information that is derived from the mass differences for different charge states of the same molecular species that are generally present in a mass spectra where molecules of differing charge states but identical mass are present, such as those formed in electro-spray ionization mass spectra. The operation of the present invention is described herein in the context of addressing space-charge effects on mass calibration for Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometry, but as will be apparent to those having skill in the art, the present invention is equally applicable to other types of instruments as well, because similar offsets in time-of-flight, sector mass spectrometers, or quadrupole ion trap data, for example, can readily be assessed using the method of the present invention. The use of the present invention with such instruments should therefore be understood to be within the scope of the present invention. The method of the present invention is also applicable in cases where ions having predictable mass differences occur, such as the case with adducts, and the present invention should be understood to include such cases.
The present invention determines the frequency shift in a way that does not require any prior knowledge of the sample, trapped ion population, or the conditions under which the measurements were made. In fact, with larger numbers of charge states, possible higher-order nonlinear frequency shifts (frequency shifts that vary across the frequency or m/z spectrum) should also be amenable to deconvolution, because subsequent pairs of charge states across the envelope could be used to effectively define the frequency shift as a function of frequency. However, the present invention described herein shows that first order, linear effects of space charge, can be corrected to provide improved mass measurement accuracy.
The present invention makes use of the fact that mass resolution sufficient to resolve isotopic peaks in electrospray source ionization (ESI) FTICR and other mass spectrometers allows definitive charge state assignment. In cases where multiple charge states are observed, as is common with electro-spray ionization, a relationship exists between the m/z of each isotopic peak for each charge state of a given species. For positively charged species resulting from protonation or other cation attachment, this relationship is defined by             (              m        /        z            )        n    =                    M        +                  n          ⁡                      (                          M              c                        )                              n        =          kB      fn      
where (m/z)n is the observed mass to charge ratio of a given peak in the isotopic envelope, n is the number of charges, M is the molecular weight, and Mc is the mass of the charge carrier, k is a proportionality constant relating m/z to the magnetic field (B) and the cyclotron frequency (fn). The first order linear shift of the observed cyclotron frequencies due to space-charge effects results in m/z values for each of the peaks being shifted from their xe2x80x9ctruexe2x80x9d position. In addition, because of the constant frequency shift and the relationship between m/z and frequency, the relationship between charge states is also affected and is observed in the xe2x80x9cdeconvolutedxe2x80x9d mass spectrum. For example, solving the above equation for M in terms of cyclotron frequency gives   M  =            n      ⁢              xe2x80x83            ⁢              kB        fn              -          n      ⁡              (                  M          c                )            
From this equation, it is clear that if all cyclotron frequencies fn are shifted by some offset Df due to space-charge effects, the observed perturbation on the deconvoluted mass, M, is charge state dependent since the quantity kB/(f1 Df) is multiplied by the charge state in the above equation. Thus, Df can be derived from the mass domain by the iterative addition or subtraction of incremental frequency shifts prior to deconvolution. The minimum error is obtained when the observed mass differences produced for different charge states are eliminated; i.e., when the optimal frequency offset due to space-charge effects has been determined.
Thus, the present invention is a method for improving the calibration of a mass spectrometer having calibration parameters by first measuring the mass to charge signal generated by ions within the mass spectrometer using the calibration parameters, then identifying a plurality of ions of equal mass having differing charge states, then adjusting the calibration parameters to cause the plurality of ions of equal mass having differing charge states to be shifted to show the same mass, and finally adjusting the measured mass to charge signal generated by the ions within the mass spectrometer utilizing the adjusted calibration parameters. In this manner, a spectrum of the ions having improved calibration may be determined. In cases where ions are present which have predictable mass differences, such as with known adducts, the present invention proceeds in an analogous manner by first measuring the mass to charge signal generated by ions within the mass spectrometer using the calibration parameters, then identifying a plurality of ions having known mass differences having differing charge states; then adjusting the calibration parameters to cause the plurality of ions having differing masses to be shifted to a relative position corresponding to the known differences in their mass, and finally adjusting the measured mass to charge signal generated by the ions within the mass spectrometer utilizing the adjusted calibration parameters.
The subject matter of the present invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. However, both the organization and method of operation, together with further advantages and objects thereof, may best be understood by reference to the following description taken in connection with accompanying drawings wherein like reference characters refer to like elements.